RESUMO
Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.
Assuntos
Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores , Poliomavírus das Células de Merkel , Células-Tronco Neoplásicas , Proteínas de Ligação a Retinoblastoma , Humanos , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/genética , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Poliomavírus das Células de Merkel/genética , Células-Tronco Neoplásicas/virologia , Células-Tronco Neoplásicas/metabolismo , Neurônios/virologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismoRESUMO
The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Carcinoma Ductal Pancreático , Linhagem da Célula , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an essential viral DNA replication protein, maintains viral persistence by interacting with host Skp1-Cullin 1-F-box (SCF) E3 ubiquitin ligase complexes, which subsequently induces LT's proteasomal degradation, restricting MCPyV DNA replication. SCF E3 ubiquitin ligases require their substrates to be phosphorylated to bind them, utilizing phosphorylated serine residues as docking sites. The MCPyV LT unique region (MUR) is highly phosphorylated and plays a role in multiple host protein interactions, including SCF E3 ubiquitin ligases. Therefore, this domain highly governs LT stability. Though much work has been conducted to identify host factors that restrict MCPyV LT protein expression, the kinase(s) that cooperates with the SCF E3 ligase remains unknown. Here, we demonstrate that casein kinase 1 alpha (CK1α) negatively regulates MCPyV LT stability and LT-mediated replication by modulating interactions with the SCF ß-TrCP. Specifically, we show that numerous CK1 isoforms (α, δ, ε) localize in close proximity to MCPyV LT through in situ proximity ligation assays (PLA) and CK1α overexpression mainly resulted in decreased MCPyV LT protein expression. Inhibition of CK1α using short hairpin RNA (shRNA) and treatment of a CK1α inhibitor or an mTOR inhibitor, TORKinib, resulted in decreased ß-TrCP interaction with LT, increased LT expression, and enhanced MCPyV replication. The expression level of the CSNK1A1 gene transcripts is higher in MCPyV-positive MCC, suggesting a vital role of CK1α in limiting MCPyV replication required for establishing persistent infection. IMPORTANCE: Merkel cell polyomavirus (MCPyV) large tumor antigen is a polyphosphoprotein and the phosphorylation event is required to modulate various functions of LT, including viral replication. Therefore, cellular kinase pathways are indispensable for governing MCPyV polyomavirus infection and life cycle in coordinating with the immunosuppression environment at disease onset. Understanding the regulation mechanisms of MCPyV replication by viral and cellular factors will guide proper prevention strategies with targeted inhibitors for MCPyV-associated Merkel cell carcinoma (MCC) patients, who currently lack therapies.
Assuntos
Antígenos Virais de Tumores , Caseína Quinase Ialfa , Poliomavírus das Células de Merkel , Proteínas Contendo Repetições de beta-Transducina , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/metabolismo , Humanos , Fosforilação , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Antígenos Virais de Tumores/metabolismo , Antígenos Virais de Tumores/genética , Interações Hospedeiro-Patógeno , Proteólise , Replicação Viral , Ligação Proteica , Antígenos Transformantes de Poliomavirus/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Infecções por Polyomavirus/virologia , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/genéticaRESUMO
BACKGROUND: Merkel Cell Carcinoma (MCC) is an aggressive skin cancer that is three times deadlier than melanoma. In 2008, it was found that 80% of MCC cases are caused by the genomic integration of a novel polyomavirus, Merkel Cell Polyomavirus (MCPyV), and the expression of its small and truncated large tumor antigens (ST and LT-t, respectively). MCPyV belongs to a family of human polyomaviruses; however, it is the only one with a clear association to cancer. METHODS: To investigate the role and mechanisms of various polyomavirus tumor antigens in cellular transformation, Rat-2 and 293A cells were transduced with pLENTI MCPyV LT-t, MCPyV ST, TSPyV ST, HPyV7 ST, or empty pLENTI and assessed through multiple transformation assays, and subcellular fractionations. One-way ANOVA tests were used to assess statistical significance. RESULTS: Soft agar, proliferation, doubling time, glucose uptake, and serum dependence assays confirmed ST to be the dominant transforming protein of MCPyV. Furthermore, it was found that MCPyV ST is uniquely transforming, as the ST antigens of other non-oncogenic human polyomaviruses such as Trichodysplasia Spinulosa-Associated Polyomavirus (TSPyV) and Human Polyomavirus 7 (HPyV7) were not transforming when similarly assessed. Identification of structural dissimilarities between transforming and non-transforming tumor antigens revealed that the uniquely transforming domain(s) of MCPyV ST are likely located within the structurally dissimilar loops of the MCPyV ST unique region. Of all known MCPyV ST cellular interactors, 62% are exclusively or transiently nuclear, suggesting that MCPyV ST localizes to the nucleus despite the absence of a canonical nuclear localization signal. Indeed, subcellular fractionations confirmed that MCPyV ST could achieve nuclear localization through a currently unknown, regulated mechanism independent of its small size, as HPyV7 and TSPyV ST proteins were incapable of nuclear translocation. Although nuclear localization was found to be important for several transforming properties of MCPyV ST, some properties were also performed by a cytoplasmic sequestered MCPyV ST, suggesting that MCPyV ST may perform different transforming functions in individual subcellular compartments. CONCLUSIONS: Together, these data further elucidate the unique differences between MCPyV ST and other polyomavirus ST proteins necessary to understand MCPyV as the only known human oncogenic polyomavirus.
Assuntos
Antígenos Virais de Tumores , Núcleo Celular , Poliomavírus das Células de Merkel , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/fisiologia , Humanos , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Núcleo Celular/virologia , Núcleo Celular/metabolismo , Animais , Ratos , Sinais de Localização Nuclear , Carcinoma de Célula de Merkel/virologia , Linhagem Celular , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/patologia , Transformação Celular Viral , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Infecções por Polyomavirus/virologiaRESUMO
Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-ß-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.
Assuntos
Antígenos Transformantes de Poliomavirus , Diferenciação Celular , Células Satélites de Músculo Esquelético , Animais , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Suínos , Antígenos Transformantes de Poliomavirus/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Proliferação de Células , Desenvolvimento Muscular , Antígenos Virais de Tumores/metabolismo , Antígenos Virais de Tumores/genética , Vírus 40 dos Símios/genéticaRESUMO
Mammalian somatic cells undergo terminal proliferation arrest after a limited number of cell divisions, a phenomenon termed cellular senescence. However, cells acquire the ability to proliferate infinitely (cellular immortalization) through multiple genetic alterations. Inactivation of tumor suppressor genes such as p53, RB and p16 is important for cellular immortalization, although additional molecular alterations are required for cellular immortalization to occur. Here, we aimed to gain insights into these molecular alterations. Given that cellular immortalization is the escape of cells from cellular senescence, genes that regulate cellular senescence are likely to be involved in cellular immortalization. Because senescent cells show altered heterochromatin organization, we investigated the implications of lamin A/C, lamin B1 and lamin B receptor (LBR), which regulate heterochromatin organization, in cellular immortalization. We employed human immortalized cell lines, KMST-6 and SUSM-1, and found that expression of LBR was upregulated upon cellular immortalization and downregulated upon cellular senescence. In addition, knockdown of LBR induced cellular senescence with altered chromatin configuration. Additionally, enforced expression of LBR increased cell proliferation likely through suppression of genome instability in human primary fibroblasts that expressed the simian virus 40 large T antigen (TAg), which inactivates p53 and RB. Furthermore, expression of TAg or knockdown of p53 led to upregulated LBR expression. These observations suggested that expression of LBR might be upregulated to suppress genome instability in TAg-expressing cells, and, consequently, its upregulated expression assisted the proliferation of TAg-expressing cells (i.e. p53/RB-defective cells). Our findings suggest a crucial role for LBR in the process of cellular immortalization.
Assuntos
Proliferação de Células , Senescência Celular , Instabilidade Genômica , Receptor de Lamina B , Receptores Citoplasmáticos e Nucleares , Humanos , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Proliferação de Células/genética , Senescência Celular/genética , Fibroblastos/metabolismo , Instabilidade Genômica/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Camundongos , Animais , Humanos , Antígenos Transformantes de Poliomavirus/metabolismo , Poliomavírus das Células de Merkel/metabolismo , NF-kappa B/metabolismo , Quinases da Família src/metabolismo , Fosfolipase C gama/metabolismo , Transdução de Sinais , Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/genética , Tirosina/metabolismoRESUMO
Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.
Assuntos
Antígenos Transformantes de Poliomavirus , Vírus 40 dos Símios , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/metabolismo , Desnaturação de Ácido Nucleico , Adenilil Imidodifosfato , Replicação do DNA , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA de Cadeia Simples , DNA Viral/genética , DNA Viral/metabolismoRESUMO
Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.
Assuntos
Antígenos Transformantes de Poliomavirus , Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Infecções Tumorais por Vírus , Humanos , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/virologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Poliomavírus das Células de Merkel/metabolismo , Fosforilação , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologia , Antígenos Transformantes de Poliomavirus/metabolismo , Transcrição GênicaRESUMO
Female SV40 C3(1) T-antigen (C3(1)/TAg) transgenic mice develop mammary tumors that are molecularly similar to human basal-like breast cancers with 100% incidence at 16 weeks of age. To determine the requirement for growth hormone (GH) signaling in these tumors, genetic crosses were used to create cohorts of female mice that were homozygous for a floxed growth hormone receptor (Ghr) gene and carried one copy each of the Rosa-Cre-ERT2 transgene and the C3(1)/TAg transgene (Ghrflox/flox; Rosa-Cre-ERT2; C3(1)/TAg+/0 mice). When the largest mammary tumor reached 200â mm3, mice were treated with tamoxifen to delete Ghr or with vehicle as a control. An additional group of Ghrflox/flox; C3(1)/TAg+/0 mice were also treated with tamoxifen when the largest mammary tumor reached 200â mm3 as a control for the effects of tamoxifen. After 3 weeks, tumors in mice in which Ghr was deleted began to shrink while vehicle and tamoxifen treatment control mouse tumors continued to grow. Pathological analysis of tumors revealed similar growth patterns and varying levels of necrosis throughout all groups. A decrease in cancer cell proliferation in Ghr-/- tumors relative to controls was observed as measured by Ki67 immunohistochemistry labeling index. These data suggest that even established C3(1)/TAg mammary tumors are dependent on the GH/IGF-1 axis.
Assuntos
Hormônio do Crescimento , Neoplasias Mamárias Experimentais , Animais , Feminino , Humanos , Camundongos , Antígenos Transformantes de Poliomavirus/genética , Proliferação de Células , Hormônio do Crescimento/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos Transgênicos , Tamoxifeno/farmacologia , Receptores de Somatostatina/genéticaRESUMO
Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT's LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT's LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Infecções Tumorais por Vírus , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Transformação Celular Neoplásica , Hiperplasia/patologia , Células de Merkel/metabolismo , Células de Merkel/patologia , Poliomavírus das Células de Merkel/genética , Camundongos , Neoplasias Cutâneas/patologiaRESUMO
Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Síndrome de Down , Fragmentos de Peptídeos/genética , Ligamento Periodontal/citologia , Telomerase/genética , Transfecção/métodos , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Síndrome de Down/genética , Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Doenças PeriodontaisRESUMO
In this study, we focused on a nuclear localization signal (NLS)-based versatile peptide vector, designed by us, combined with electroporation (EP) to establish an efficient gene delivery system to non-dividing or slow growing dendritic cells. We determined the intranuclear transport, gene expression, and cell viability in JAWS II mouse dendritic cells transfected with the green fluorescent protein (GFP) expression plasmid DNA alone (naked pEGFP); positive charged complex of NLS derivative STR-CH2SV40H2C, and pEGFP (binary complex); or negative charged complex of the binary complex with a biocompatible polyanion, γ-polyglutamic acid (ternary complex) combined with or without EP application. Although the binary complex showed higher nuclear transport and GFP expression in the absence of EP than those for naked pEGFP, the combination of EP significantly decreased the cell viability and did not improve the efficiency of compared gene expression. However, in the ternary complex, the intranuclear transport and GFP expression efficiency were significantly higher than those of naked pEGFP and the binary complex when combined with EP, and there was no decrease in cell viability. The results suggest that polyanion-coated ternary complex with EP is useful for non-viral gene delivery system into non-dividing cells for ex vivo gene/cell therapy.
Assuntos
Núcleo Celular , Células Dendríticas , Eletroporação , Técnicas de Transferência de Genes , Terapia Genética , Sinais de Localização Nuclear , Polímeros , Animais , Antígenos Transformantes de Poliomavirus , Sobrevivência Celular , DNA , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoterapia , Camundongos , Neoplasias/terapia , Oligopeptídeos , Plasmídeos , Ácido Poliglutâmico/análogos & derivados , TransfecçãoRESUMO
Cell immortalization enables us to expand the cultured cell infinitely. However, the process of immortalization sometimes changes the nature of the original cell. In this study, we established immortalized embryonic fibroblasts with oncogenic SV40T and human papilla virus-derived E6E7, combinational expression of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomerase reverse transcriptase (TERT) from identical primary wild-type human embryonic fibroblasts (HE16). After the establishment of immortalized cells, we compared the details of chromosome condition with the G-banding and Q-banding methods. There is no example of detailed analysis so far about chromosome abnormalities, such as trisomy, ring chromosome, reciprocal translocation, and dicentric chromosomes. The detailed chromosome analysis revealed that immortalized cells with SV40T and E6E7 showed intensive chromosome abnormalities, such as gain or loss of the chromosomes all through the genome. Furthermore, we detected that the incidence of chromosome abnormities in the immortalized cell with the combinational introduction of R24C mutant of CDK4, cyclin D1, and TERT is almost identical to that of wild-type cell. Furthermore, short tandem repeat analysis demonstrated that the origin of K4DT cell is primary HE16. These results showed that cellular immortalization with CDK4, cyclin D1, and TERT is more advantageous in keeping the chromosome's original condition than oncogenic immortalization methods.
Assuntos
Cromossomos Humanos , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Fibroblastos/citologia , Telomerase/genética , Antígenos Transformantes de Poliomavirus/genética , Ciclo Celular/genética , Linhagem Celular Transformada , Bandeamento Cromossômico , Humanos , Cariotipagem , Proteínas Oncogênicas Virais/genéticaRESUMO
Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/terapia , Metaboloma/efeitos dos fármacos , Proteínas do Tecido Nervoso/fisiologia , Adulto , Idoso , Animais , Antígenos Transformantes de Poliomavirus/genética , Meios de Cultivo Condicionados , Feminino , Glicólise/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Receptores de Lipopolissacarídeos/análise , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Quimera por Radiação , Serina-Treonina Quinases TOR/fisiologia , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/química , Carga TumoralRESUMO
Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast carcinogenesis using the transgenic MMTV-PyMT mouse mammary carcinoma model. We show here for the first time that the inactivation of Col15a1 in mice leads to changes in the fibrillar tumour matrix and to increased mammary tumour growth. ColXV is expressed by myoepithelial and endothelial cells in mammary tumours and is lost from the ductal BM along with the loss of the myoepithelial layer during cancer progression while persisting in blood vessels and capillaries, even in invasive tumours. However, despite the absence of anti-angiogenic restin domain, neovascularisation was reduced rather than increased in the ColXV-deficient mammary tumours compared to controls. We also show that, in robust tumour cell transplantation models or in a chemical-induced fibrosarcoma model, the inactivation of Col15a1 does not affect tumour growth or angiogenesis. In conclusion, our results support the proposed tumour suppressor function of ColXV in mammary carcinogenesis and reveal diverse roles of this collagen in different cancer types.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Colágeno/deficiência , Matriz Extracelular/metabolismo , Deleção de Genes , Neoplasias Mamárias Animais/patologia , Vírus do Tumor Mamário do Camundongo/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/patologia , Proliferação de Células , Colágeno/genética , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Fibrossarcoma/patologia , Fibrose , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/patologia , Células Estromais/ultraestrutura , Análise de SobrevidaRESUMO
RATIONALE: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. AIM: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. METHODS AND RESULTS: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor ß3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. CONCLUSION: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.
Assuntos
Pericárdio/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Doxiciclina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Gânglios Simpáticos/citologia , Gânglios Simpáticos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Modelos Biológicos , Neuritos/fisiologia , Comunicação Parácrina/efeitos dos fármacos , Pericárdio/citologia , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
A high-fat diet (HFD) and obesity are risk factors for many diseases including breast cancer. This is particularly important with close to 40% of the current adult population being overweight or obese. Previous studies have implicated that Mediterranean diets (MDs) partially protect against breast cancer. However, to date, the links between diet and breast cancer progression are not well defined. Therefore, to begin to define and assess this, we used an isocaloric control diet (CD) and two HFDs enriched with either olive oil (OOBD, high in oleate, and unsaturated fatty acid in MDs) or a milk fat-based diet (MFBD, high in palmitate and myristate, saturated fatty acids in Western diets) in a mammary polyomavirus middle T antigen mouse model (MMTV-PyMT) of breast cancer. Our data demonstrate that neither MFBD or OOBD altered the growth of primary tumors in the MMTV-PyMT mice. The examination of lung metastases revealed that OOBD mice exhibited fewer surface nodules and smaller metastases when compared to MFBD and CD mice. These data suggest that different fatty acids found in different sources of HFDs may alter breast cancer metastasis.
Assuntos
Neoplasias da Mama/patologia , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/toxicidade , Ácidos Graxos/toxicidade , Neoplasias Pulmonares/secundário , Leite/toxicidade , Ração Animal , Animais , Antígenos Transformantes de Poliomavirus , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Azeite de Oliva/toxicidade , Medição de Risco , Fatores de Risco , Carga Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We describe our initial studies in the development of an orthotopic, genetically defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Transformação Celular Neoplásica/genética , Células Epiteliais/patologia , Genes ras , Ductos Pancreáticos/citologia , Neoplasias Pancreáticas/patologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Divisão Celular , Linhagem Celular Transformada , Células Epiteliais/transplante , Xenoenxertos , Masculino , Camundongos , Camundongos Nus , Modelos Animais , Mutação de Sentido Incorreto , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Mutação Puntual , SuínosRESUMO
Cellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.